SOME  EXPERIMENTS  ON  THE  DEVELOPINQ' .V 
VESICLE  OF  THE  TADPOLE  WITH  RELATION'*' ••  K 

TO  EQUILIBRATION  / 


•  •  •• 
•  ••• 


»  •  •  • 


•  .  •  •  •  «  < 

r  •  •  * 

• .  •  •  •  ••  • 

•••  • 

•  •••••  »• 

» •  ••  •*  • 


By 

George  L.  Streeter,  M.D. 


reprinted  from 

THE  JOURNAL  OF  EXPERIMENTAL  ZOOLOGY 

Volume  III 
No.  4 


if- 1 

■4, 


'T/I 


I. 


BALTIMORE,  MD.,  U.  S.  A 
December,  1906 


r 


> 


V 


From  the  Anatomical  Laboratory  of  the  Johns  Hopkins  University 


SOME  EXPERIMENTS  ON  THE  DEVELOPING  EAR 
VESICLE  OF  THE  TADPOLE  WITH  RELATION  , 

TO  EQUILIBRATION* 

BY 

GEORGE  L.  STREETER,  M.D. 

Associate,  Wistar  Institute  of  Anatomy 
With  Twelve  Figures 

The  eventual  object  of  the  experiments  reported  in  the  following 
paper  was  the  rearing  of  some  tadpoles  which  had  been  deprived 
of  their  auditory  vesicle  and  acoustic  ganglion,  either  on  one  side 
alone  or  on  both  sides;  that  is  to  say,  an  artificial  production  of  a 
unilateral  and  bilateral  absence  of  the  acoustic  apparatus.  This 
was  done  in  the  expectation  that  it  might  be  possible  to  trace  the 
central  acoustic  path,  in  this  new  way,  and  perhaps  throw  further 
light  upon  its  course  and  relations.  The  absence  of  these  sense 
organs,  however,  produced  such  definite  abnormalities  in  the 
behavior  of  the  growing  larvae  and  in  the  development  of  their  swim¬ 
ming  abilities  that  it  became  at  once  apparent  that  I  was  dealing 
with  valuable  evidence  in  respect  to  their  function  and  its  bearing 
on  the  mechanism  of  equilibrium.  It  is,  therefore,  deemed  advisable 
to  restrict  the  following  paper  to  the  physiological  features  of  these 
experiments,  and  reserve  the  study  of  the  central  nervous  system 
of  the  reared  specimens  for  a  later  communication. 

What  we  already  know  concerning  the  function  of  the  vertebrate 
ear  is  based  principally  on  experimental  sectioning  or  stimula¬ 
tion  of  the  semicircular  canals,  or  the  nerves  to  their  ampullae,  in 
adult  birds  and  fishes.^ 

^Read  in  part  before  the  Section  of  Anatomy  of  the  British  Medical  Association,  at  the  meeting  held 
in  Toronto,  August  21-25,  ^906. 

^For  experimental  work  on  fishes  we  are  for  the  most  part  indebted  to  Lee  (’93  and  ’98)  and  Lyon 
(’00),  both  of  whom  carried  on  their  experiments  at  the  Woods  Hole  Laboratories.  Further  work  on 
fishes  has  just  been  completed  at  the  same  place  by  Professor  Parker,  whose  paper  I  am  told  is  now  in 
press  and  will  appear  in  the  Bulletin  of  the  U.  S.  Fisheries  Bureau.  An  abstract  of  part  of  his 
work  was  read  before  the  American  Zoological  Society  (Parker,  ’05).  A  voluminous  literature  exists 
concerning  experiments  on  higher  vertebrates,  particularly  the  pigeon,  but  it  need  not  be  considered 
here. 

The  Journal  of  Experimental  Zoology,  vol.  111,  no.  4. 

u  t  ^  SI 


544 


George  L.  Streeter 


The  fact  that  it  is  possible  to  experiment  on  the  embryo  and  to 
produce  at  will  practically  a  congenital  absence  of  thisorgan,  besides 
serving  as  a  control  over  the  experiments  on  adult  animals,  intro¬ 
duces  a  direct  advantage  both  as  regards  the  ease  with  which  the 
operation  is  performed  and  as  regards  its  completeness  and  per¬ 
manence  and  freedom  from  injury  of  adjoining  structures,  the  latter 
point  being  of  particular  importance  to  those  who  are  still  in  doubt 
.  as  to  how  much  is  due  in  the  experiments  on  adults  to  injuries  and 
stimulationsassociated  with  the  operation  and  how  much  is  purely 
the  result  of  the  cessation  of  the  stimuli  which  normally  originate  in 
the  labyrinth.  Furthermore,  since  the  labyrinth  is  removed  during 
the  early  formative  period  at  a  time  when  it  may  be  presumed  that 
the  various  organs  possess  their  greatest  adaptability,  it  will  be  seen 
that  such  embryonic  interference  affords  a  most  complete  test 
of  the  power  of  functional  compensation  on  the  part  of  other 
organs. 

Behavior  of  Normal  Tadpoles 

In  analyzing  the  behavior  of  operated  specimens  it  was  found 
necessary  to  make  a  preliminary  study  of  control  tadpoles,  in  order 
to  determine  the  normal  development  of  motor  reflexes  and  their 
coordination  and  the  consequent  establishment  of  equilibrium. 
This  was  done  by  removing  the  larvae  from  their  gelatinous  cap¬ 
sule  shortly  after  fertilization  and  following  their  development  in 
tap  water.  In  this  way  it  was  seen  that  in  the  process  of  learning 
to  swim  they  pass  through  three  periods,  which  may  be  named  as 
follows : 

1.  Stage  of  non-motility,  first  three  days. 

2.  Stage  of  spinal  reflexes,  fourth  to  sixth  days. 

3.  Stage  of  equilibrium,  sixth  day  to  maturity. 

The  first  stage,  with  a  favorable  temperature,  lasts  from  the 
time  of  fertilization  to  the  third  or  fourth  day.  During  this  time 
the  larvae,  aside  from  the  movement  due  to  cilia,  lie  motionless  on 
their  side  on  the  bottom  of  the  dish  and  do  not  respond  to  stimuli. 
The  second  stage  begins  at  the  time  when  they  first  respond  to 


Experiments  on  the  Developing  Ear  Vesicle 


545 


mechanical  stimuli  by  flexion  of  the  body  and  taild  These  reac¬ 
tions  consist  of  simple  motor  reflexes  at  first,  but  they  soon  become 
combined  and  coordinated  so  that  by  a  series  of  such  body  flexions 
they  are  able  to  wiggle  rapidly  forward  on  the  bottom  of  the  dish. 
This  manner  of  progression  evidently  consists  entirely  of  spinal 
cord  reflexes  and  is  not  controlled  by  higher  centers.  In  order  to 
perform  it,  it  is  necessary  for  the  tadpoles  to  touch  the  bottom  or 


Fig.  I,  Outline  drawing  of  normal  tadpole  (Rana  sylvatica)  of  the  second  stage  or  stage  of  spinal 
reflexes.  Enlarged  8  diameters. 

Fig.  2,  Outline  drawing  of  normal  tadpole  (Rana  sylvatica)  at  the  beginning  of  the  third  stage.  This 
specimen  had  the  power  of  equilibration,  although  sections  of  the  ear  vesicle  showed  that  the 
development  of  the  semicircular  canals  was  not  yet  complete.  Enlarged  8  diameters. 

side  of  the  dish;  when  they  are  driven  up  into  the  free  water  with 
a  pipette,  where  there  is  no  contact  with  solid  objects,  they  make 
no  effort  at  movement,  but  sink  inertly  to  the  bottom;  on  striking 
the  bottom  they  run  forward  again.  The  third  stage  begins  when 
they  are  first  able  to  move  freely  about  without  touching  solid 

^  I  have  been  informed  by  Dr.  R.  G.  Harrison  that  it  is  just  at  this  time  that  the  motor  nerve 
roots  make  their  appearance,  and  this  may  determine  the  onset  of  the  second  stage.  According  to 
his  observations  the  power  of  muscle  contraction  follows  almost  immediately  after  the  development 
of  the  motor  roots;  but  it  never  precedes  their  development,  as  is  maintained  by  some.  He  has 
found  the  motor  root  present  in  specimens  that  had  not  yet  moved. 


54^  George  L.  Streeter 

objects.  At  this  time  a  new  control  over  their  movements  is  devel¬ 
oped,  in  virtue  of  which  they  become  able  to  leave  the  bottom  of  the 
dish  and  swim  up  into  free  water  with  maintenance  of  what  may 
then  be  called  equilibrium.  The  form  of  the  tadpole  during  the 
latter  part  of  the  first  stage  is  shown  in  Fig.  3.  The  second  and 
third  stages  are  shown  in  Figs,  i  and  2. 

The  correlation  between  the  histological  development  of  the 
labyrinth  and  the  development  of  the  power  of  equilibrium  was 
studied  by  selecting  specimens  of  the  second  and  the  beginning  of 
the  third  stages,  carefully  noting  their  behavior,  and  then  cutting 
them  in  serial  sections.^ 

From  these  series  it  could  be  seen  that  shortly  before  the  animal 
enters  the  stage  of  equilibrium  the  labyrinth  consists  of  a  closed 
epithelial  sac  incompletely  subdivided  into  compartments  and 
possessing  differentiated  nerve  endings  which  are  connected  with 
the  brain  by  the  acoustic  nerve  and  ganglion.  That  at  least  one 
such  apparatus  is  essential  for  equilibrium  will  be  seen  when  I 
describe  the  behavior  of  tadpoles  that  have  been  completely 
deprived  of  the  same.  As  regards  the  semicircular  canals  it  is  a 
different  matter;  they  can  already  be  seen  in  the  process  of 
development,  but  are  not  completely  pocketed  off  until  after  equili¬ 
brium  is  already  established.  Consequentlythe  semicircular  canals 
as  such  are  not  an  essential  factor  in  equilibration. 

Method  of  Operation 

Larvap  of  Rana  sylvatica  measuring  about  3  mm.  long  were 
selected  as  being  most  suitable  for  the  operation.  Their  general 
form  at  this  time  is  shown  in  Fig.  3.  There  is  a  distinct  tail  bud, 
and  on  the  head  the  eminences  caused  by  the  optic  cup  and  head 
ganglia  are  visible.  The  structure  that  is  to  form  the  future  laby¬ 
rinth  is  situated  just  dorsal  to  the  ganglionic  eminence  and  is  shown 

^  The  correlation  between  the  histogenesis  of  organs  and  the  development  of  their  functional  activity 
forms  a  fruitful  field  which  has  been  explored  by  comparatively  few  investigators.  It  may  be  approached 
both  through  ontogeny  and  phylogeny.  Prentiss  (’oi)  by  this  means  worked  out  important  facts 
regarding  the  crustacean  otocyst.  Many  details  concerning  the  verte  brate  ear  which  do  not  belong  to 
the  scope  of  the  present  paper  could  doubtless  be  learned  in  the  same  way. 


Experiments  on  the  Developing  Ear  V esicle 


547 


in  Fig.  3  by  the  mark  +.  It  consists  of  a  cup-shaped  mass  of 
cells  (auditory  cup)  which  have  differentiated  themselves  from 
the  deeper  layer  of  epidermis,  and  are  just 
in  the  process  of  closing  in  at  the  edges  to 
form  the  completed  ear  vesicle.  In  size 
this  ear  cup  or  ear  vesicle  is  about  one- 
half  that  of  the  optic  cup. 

For  performing  the  operation  it  is  not 
necessary  to  anesthetize  the  specimen  as 
it  is  still  in  the  non-motile  stage  and  does 
not  respon d  to  stimulation.  After  remov¬ 
ing  the  larva  from  its  gelatinous  capsule  it 

and  an  incision  made  near  the  place  indi¬ 
cated  in  Fig.  3.  The  edge  of  the  incision 
is  then  raised  a  little  and  the  auditory  cup 
is  picked  out  with  a  needle.  After  a  little 
practice  one  learns  to  make  the  incision  directly  at  the  edge  of  the 
cup  so  that  it  comes  away  easily  and  intact,  resembling  somewhat 
a  thimbleberry.  Lying  just  in  front  of  it  is  the  acoustic  ganglion 
which  is  not  so  sharply  outlined.  This  is  also  removed  and, 
in  order  to  make  sure  that  it  is  all  taken  out,  the  surrounding 
mesoderm  is  cleaned  out  as  far  in  as  the  brain.  Where  but  one 
vesicle  is  to  be  removed  the  operation  is  then  complete,  and  the 
specimen  is  left  to  proceed  in  its  development.  The  wound 
immediately  closes  of  itself  and  heals  in  the  course  of  a  few  hours 
leaving  no  trace  of  the  operation.  Where  both  sides  are  operated 
on,  the  same  procedure  is  carried  out  on  both  sides.  The  ear 
vesicle  never  regenerates  following  complete  removal. 

The  ear  vesicle  was  removed  on  one  side  from  thirty  specimens 
and  on  both  sides  from  twenty  specimens.  The  animals  were  then 
kept  under  observation  and  their  behavior  recorded  through  the 
whole  larval  period  and  until  the  completion  of  metamorphosis. 
The  following  notes  were  selected  from  these  records. 


is  placed  under  a  binocular  microscope 


Fig.  3.  Outline  drawing  of  Rana 
sylvatica  at  the  time  suitable  for 
operation,  just  at  the  end  of  the  non- 
motile  stage.  The  tail  bud  is  present 
and  on  the  head  are  seen  the  emi¬ 
nences  due  to  the  optic  cup  and  head 
ganglia.  Above  the  latter  is  the 
point  of  operation  shown  by  a  cross. 
Enlarged  8  diameters. 


54^  George  L.  Streeter 

Removal  of  One  Ear  V esicle 

Twenty-four  hours  after  operation :  Specimens  are  5.5  mm.  long 
and  show  presence  of  gill  buds.  In  appearance  and  behavior^  no 
difference  can  be  detected  between  them  and  normal  tadpoles. 
They  lie  on  their  side  and  on  stimulation  flex  their  body,  but  make 
no  attempt  at  swimming. 

Forty-eight  hours  after  operation :  Specimens  are  7  mm.  long, 
gills  are  branched  and  the  blood  can  be  seen  circulating  through 
them.  In  appearance  and  behavior  they  still  show  no  departure 
from  that  seen  in  normal  control  specimens.  While  at  rest  they 
lie  on  theii  side.  On  stimulation  (sunlight,  jarring  the  dish,  or 
touching  with  needle)  by  a  rapid  flexion  of  the  body  and  tail  from 
side  to  side  they  swim  forward,  5-10  cm.,  on  the  bottom  of  the 
dish  in  a  straight  or  slightly  curved  line,  and  then  come  to  rest  on 
their  side,  and  remain  so  until  a  new  stimulation  excites  another 
such  excursion.  Their  course  is  directed  either  by  the  side  or 
bottom  of  the  dish.  When  forced  up  into  free  water  the  flexions 
stop  and  they  sink  inertly  to  the  bottom. 

Third  day  after  operation:  Specimens  average  about  8  mm. 
long,  abdominal  epidermis  differentiated  from  that  of  the  dorsal 
parts  of  the  body  by  being  less  pigmented.  Appearance  and  be¬ 
havior  is  still  practically  normal.  They  begin  to  show  a  tendency 
to  assume  the  upright  position  while  at  rest,  but  no  great  impor¬ 
tance  can  be  attached  to  this  feature  as  throughout  the  early  days 
of  the  tadpole  period,  preserved  specimens  lie  in  the  same  positions 
as  living  ones.  Their  posture  in  water  may  be  entirely  determined 
by  their  body  proportions.  Their  movements  remain  of  the  spinal 
cord  type  seen  on  the  previous  day,  the  response  being  more  prompt. 

Fourth  day  after  operation:  Specimens  9-9.5  mm.  long.  In 
appearance  the  operated  specimens  are  the  same  as  the  normal 
ones,  but  in  behavior  they  present  a  difference.  The  normal  ones 
still  confine  their  movements  to  the  bottom»or  side  of  the  dish; 
when  stirred  up  into  free  water,  though  most  of  them  still  roll  about 
inertly,  some  of  them  are  able  to  maintain  a  direct  course.  On  the 
other  hand  the  operated  ones,  as  soon  as  they  are  driven  from  the 
bottom,  swim  in  a  spiral  or  circular  manner  as  shown  in  the 


Experiments  on  the  Developing  Ear  V esicle 


549 


accompanying  Fig.  4.  The  tendency  is  to  swim  with  the  operated 
side  under,  and  in  the  rolling  movements  around  the  long  axis  of 
the  body  it  is  from  the  operated  side  under  to  the  opposite.  When 
these  same  specimens  touch  the  bottom  they  are  able  to  direct  their 
course  as  on  the  previous  two  days.  Evidently,  a  functional  union 
is  normally  established  at  about  this  time  between  the  ear  vesicle 
and  the  spinal  cord  reflex  centers,  upon  which  the  individual  is 
dependent  for  maintafning  its  position  in  free  water,  and  it  is  not 
until  this  occurs  that  the  removal  of  the  ear  vesicle  causes  any 
symptoms. 


Fig.  4.  Sketch  showing  three  typical  swimming  movements  made  by  specimens  on  the  fourth  day 
after  removal  of  their  left  ear  vesicle. 

Sixth  day  after  operation:  Specimens  about  12  mm.  long,  and 
have  commenced  to  nibble  at  food  and  pass  faeces.  The  charac¬ 
teristic  movements  which  first  appeared  on  the  fourth  day  have 
become  stronger  and  stand  out  in  more  marked  contrast  to  the 
behavior  of  normal  specimens  which  at  this  time  can  swim  easily 
up  into  free  water  with  accurate  maintenance  of  equilibrium. 

Seventh  day  after  operation:  The  operated  specimens  show 
distinct  improvement  in  swimming  ability;  many  of  them  are  now 
able  to  maintain  a  fairly  direct  course  in  free  water,  but  on  excita¬ 
tion  they  renew  the  spirals  and  circles  which  characterized  the 
fourth,  fifth  and  sixth  days. 


550 


George  L.  Streeter 


Eighth  day  after  operation:  nearly  all  the  specimens  now 
swim  freely  and  directly  in  all  parts  of  the  water,  and  irregularity 
of  swimming  is  only  elicited  by  excitement. 

Tenth  day  after  operation:  Swimming  is  practically  normal. 
Their  movements  are  under  such  control  as  to  enable  them  to  sup¬ 
port  themselves  in  free  water  and  nibble  at  floating  stems  and 
leaves.  It  can  be  seen,  however,  that  in  swimming  they  lean 


Fig.  5.  Photograph  of  a  frog  whose  left  ear  vesicle  was  removed  when  a  tadpole  3  mm.  long.  The 
only  asymmetry  noticeable  is  the  absence  of  the  ear  elevation  on  the  left  side  normally  caused  by  the 
labyrinth  and  its  cartilaginous  capsule;  the  lateral  line  on  that  side  is  straight  from  the  eye  back,  while 
on  the  right  or  normal  side  it  is  deflected.  The  posture  is  normal.  Enlarged  3^  diameters. 


slightly  toward  the  operated  side,  a  sympton  which  persists  through¬ 
out  their  larval  period. 

Twelfth  day  after  operation:  Specimens  are  normal  as  regards 
size,  nourishment,  and  symmetry,  except  for  the  absence  on  the 
operated  side  of  the  elevation  which  is  caused  normally  by  the 
labyrinth  and  its  cartilaginous  capsule.  In  behavior  they  differ 
from  the  normal  only  in  the  slight  leaning  toward  the  operated  side 


Experiments  on  the  Developing  Ear  Vesicle  55^ 

and  a  momentary  loss  of  equilibrium  which  can  be  elicited  by 
excitement. 

Three  months  after  operation:  The  specimens  passed  through 
a  normal  metamorphosis  at  the  end  of  the  third  month.  A  photo¬ 
graph  made  of  one  of  them  a  few  days  after  the  completion  of  the 
process  is  shown  in  the  accompanying  Fig.  5. 

As  long  as  they  continued  as  swimming  tadpoles  the  slight  lean¬ 
ing  toward  the  operated  side  persisted  and  it  was  possible  through 
excitement  to  cause  a  momentary  disturbance  in  equilibrium,  but 
the  latter  became  gradually  more  difficult  to  demonstrate.  As 
soon  as  they  commenced  to  make  use  of  their  legs  the  character 
of  the  swimming  changed;  it  then  became  a  series  of  leg  strokes 
instead  of  the  sinuous  flexions  of  the  body  and  tail.  After  that  it 
was  no  longer  possible  to  detect  the  leaning  toward  the  operated 
side;  both  when  swimming  and  when  at  rest  their  behavior  was  to 
all  appearance  normal.  When  taken  out  of  water  they  jumped 
normally  and  came  to  rest  in  a  normal  posture.  When  turned 
over  on  their  backs  they  righted  themselves  promptly. 

The  fact  that  the  slight  disturbance  of  equilibrium,  which  could 
be  still  detected  in  the  tail-swimming  tadpole,  could  no  longer  be 
seen  in  the  leg-swimming  frog,  a  change  completed  within  four  or 
five  days,  probably  does  not  signify  the  cure  of  the  condition,  but 
rather  that  under  the  latter  circumstances  a  slight  defect  is  more 
difficult  to  recognize.  The  corollary  of  this  would  be  that  equili¬ 
brium  in  the  swimming  tadpole  is  a  more  delicately  balanced 
mechanism  than  in  the  kicking  and  jumping  frog. 


Removal  of  Both  Ear  Vesicles 

During  the  first  three  days  after  the  operation  the  appearance 
and  behavior  of  these  specimens  are  the  same  as  seen  in  the  normal 
ones,  and  in  those  from  which  one  ear  vesicle  was  removed.  The 
response  to  stimuli  is  perhaps  a  trifle  less  prompt,  but  otherwise 
they  could  not  be  distinguished  one  from  the  other. 

Fourth  day  after  operation :  It  was  seen  that  in  one-sided  oper¬ 
ations  the  specimens  commenced  about  this  time  to  make  excur- 


552 


George  L.  Streeter 


sions  into  free  water,  and  in  doing  it  they  departed  from  the  nor¬ 
mal  by  swimming  in  spirals  and  circles.  Tadpoles  with  both  ear 
vesicles  taken  out  make  no  such  excursions  and  show  decidedly* 
less  activity.  Occasionally  they  flex  their  body  and  tail  from  side 
to  side  producing  a  snapping  effect  which  does  not  result  in  any 
forward  progress.  Like  the  other  specimens  they  are,  however, 
able  to  wiggle  along  in  contact  with  the  side  and  bottom  of  the 
dish.  * 

Seventh  day  after  operation:  The  specimens  are  smaller  and 
are  retarded  in  development  as  compared  with  the  normal  and 
one-sided  specimens.  They  are,  however,  symmetrical  in  form 
and  are  normal  as  regards  the  appearance  and  movements  of  the 
eyes,  mouth,  heart  and  intestine.  They  are  decidedly  less  active 
and  stimuli  produce  irregular  attempts  at  swimming,  sometimes 
somewhat  spiral  in  character  but  usually  nothing  more  than  a 
series  of  awkward  flexions  of  the  body.  These  flexions  also 
occasionally  occur  with  no  apparent  stimulus.  They  make  a 
partially  successful  effort  at  nibbling  on  the  bottom  of  the  dish. 

Twelfth  day  after  operation:  Absolutely  no  improvement  in 
swimming;  any  attempt  at  it  results  in  a  series  of  somersaults, 
they  throw  their  body  up  into  the  water  and  then  promptly  sink 
to  the  bottom  in  almost  any  position.  When  at  rest,  they  lie  on 
their  side,  back,  or  normally  on  their  belly,  depending  apparently 
on  whether  their  intestine  is  filled  with  sand,  etc.,  to  properly 
balance  the  body.  The  intestine  is  very  apt  to  be  empty  because 
of  the  difficulty  they  experience  in  feeding.  They  do  not  wiggle 
along  on  the  bottom  as  well  as  they  did  on  the  fourth  and  fifth 
days. 

Two  months  after  operation:  The  specimens  could  not  be 
carried  much  beyond  this  point,  the  difficulty  apparently  being 
starvation  from  inability  to  wander  around  and  collect  food. 
Perhaps  also  the  respiration  was  involved,  for  they  were  unable 
to  go  to  the  surface  for  oxygen  as  the  normal  tadpole  does. 

In  behavior  they  show  no  improvement.  For  the  most  part  they 
lie  stiff  and  inert  in  various  positions  on  the  bottom,  and  their 
occasional  attempts  at  swimming  have  never  developed  into  anything 
more  successful  than  was  described  on  the  seventh  and  twelfth 


Experiments  on  the  Development  Ear  Vesicle 


553 


days  after  operation.  Their  appearance  departs  from  the  nor¬ 
mal  principally  in  the  small  contracted  character  of  the  abdominal 
region.  In  volume  they  are  about  one-third  as  large  as  the  noimal 
specimen,  varying  from  2.5  to  4  cm.  in  length.  They  have  a  hind 
leg  bud  2.5  to  3  mm.  long.  As  some  of  them  commenced  to 
die  at  this  time  the  rest  were  put  in  preserving  fluids  for  micro- 
scopial  purposes. 

A  summary  of  the  above  notes  on  the  operated  iindividuals  may 
perhaps  be  best  formulated  by  making  the  following  comparison 
with  the  three  stages  of  normal  behavior. 

First  stage:  The  operation  was  performed  during  the  latter 
part,  while  the  animals  were  still  non-motile. 

Second  stage:  During  this  period  they  behave  exactly  like 
normal  specimens,  both  those  having  one  vesicle  removed  and 
those  that  have  been  deprived  of  both  vesicles.  They  respond  to 
stimuli  and  learn  to  wiggle  along  in  contact  with  the  bottom  of  the 
dish  in  the  normal  manner. 

Third  stage:  It  is  at  the  beginning  of  this  period  that  they 
depart  from  the  normal.  It  can  be  plainly  seen  from  their  conduct 
that  something  has  happened  to  that  controlling  influence  from' 
above,  which  they  require  in  order  to  leave  the  bottom  and  to  swim 
and  maintain  their  position  in  free  water.  In  case  but  one  ear 
vesicle  is  gone  they  swim  in  spirals,  circles,  or  straight  while  roll¬ 
ing  around  their  long  axis.  This,  however,  lasts  only  a  few  days 
and  then  it  is  gradually  overcome.  From  then  on  they  swim 
almost  perfectly;  there  maybe  a  slight  tilting  toward  the  operated 
side  and  on  excitement  a  momentary  loss  of  equilibrium,  but  this 
would  only  be  seen  on  careful  examination.  It  is  a  different  mat¬ 
ter  where  both  labyrinths  are  absent;  the  animals  in  that  case  are 
completely  and  permanently  incapacitated  for  swimming.  There 
is  no  apparent  sense  of  equilibrium  and  they  never  develop  any. 
The  animals  were  kept  alive  about  two  months,  at  the  end  of 
which  time  their  movements  were  as  irregular  as  at  the  beginning. 

Transplantation  of  Ear  Vesicle  After  Bilateral  Removal 

From  the  above  experiments  it  became  evident  that  a  tadpole 
having  but  one  labyrinth  proceeds  in  its  general  growth  and 


554 


George  L.  Streeter 


develops  swimmingabilities  about  as  well  as  the  normal  animal;  but 
specimens  deprived  of  both  ear  vesicles  never  learn  to  swim  and 
never  develop  any  sense  of  equilibrium.  The  next  step  was  to 
see  if  it  would  be  possible  to  remove  both  vesicles  and  atthe  same 
time  transplant  one  of  them  into  a  new  position,  having  in  mind 
the  successful  results  obtained  by  Lewis  (’04)  in  transplantation  of 
the  optic  cup. 

After  that  operation  if  the  tadpole  succeeded  in  developing 
equilibrium  and  the  power  of  swimming  then  it  would  prove  that 
a  transplanted  ear  vesicle  could  establish  new  connections  with  the 
central  nervous  system  and  develop  its  normal  functions;  the  ship 
would  simply  be  sailing  with  its  compass  set  up  in  a  different  place. 


Fig.  6.  Tadpole  showing  elevation  in  front  of  eye  caused  by  the  transplanted  left  ear  vesicle,  the 
right  ear  vesicle  having  been  entirely  removed.  Drawing  made  three  months  after  operation.  Enlarged 
diameters.  , 


The  operation  was  one  that  could  be  performed  without  diffi¬ 
culty.  A  tadpole  about  3  mm.  long  is  selected  and  the  ear 
vesicle  taken  out  on  one  side  in  the  manner  described  above.  The 
specimen  is  then  turned  over  and  the  opposite  ear  vesicle  is 
uncovered  and  loosened  from  the  epidermis.  Before  actually 
removing  it  a  straight  incision  is  made  with  scissors  or  needles  in 
front  of  the  eye  and  a  pocket  is  created  by  gently  spreading  the  sub¬ 
jacent  mesoderm  apart  until  the  brain  is  exposed.  The  loosened 
ear  vesicle  is  then  lifted  from  its  natural  place  and  slipped  into 
this  pocket.  If  the  incision  is  carefully  made  the  edges  of  the 
wound  close  at  once  and  on  the  following  day  there  is  no  trace  of 
the  operation  left.  Nine  operations  of  this  kind  were  made  and 
seven  of  the  tadpoles  successfully  reared.  While  they  were  grow¬ 
ing  it  could  be  seen  from  a  surface  view  that  the  transplanted 
vesicle  was  developing  and  causing  a  corresponding  elevation  in 


555 


Experiments  on  the  Developing  Ear  V esicle 

front  of  the  eye.  A  sketch  of  one  of  these  at  the  end  of  the  third 
month  is  shown  in  Fig.  6. 

Their  behavior  during  the  first  week  following  the  operation  was 
identical  with  that  of  specimens  deprived  of  both  vesicles  as  was 
to  be  expected.  Toward  the  fifth  and  sixth  days  they  could 
make  progress  while  touching  the  side  or  bottom  of  the  dish,  but 
any  attempt  at  swimming  in  free  water  resulted  only  in  irregular 
flexions  of  the  body  and  somersaults.  It  was  hoped  that  the  trans¬ 
planted  vesicle  might  then  begin  to  function  and  make  it  possible 
for  them  to  perceive  their  position  while  in  free  water,  but  this  did 
not  occur.  They  continued  to  behave  in  all  respects  like  tadpoles 
having  no  labyrinth  and  never  gave  evidence  of  possessing  any 
trace  of  equilibrium.  * 

At  the  end  of  the  third,  fourth  and  twelfth  weeks  specimens 
were  killed  in  preserving  fluid  and  prepared  in  serial  sections. 
Examination  of  the  sections  showed  that  in  six  out  of  the  seven 
specimens  which  were  cut,  the  transplanted  vesicle  had  developed 
to  a  greater  or  less  extent,  and  it  was  these  vesicles  that  formed  the 
surface  elevations  that  had  been  macroscopically  visible  in  front 
of  the  tadpole’s  eyes.  Graphic  reconstructions  of  them  are  repre¬ 
sented  in  Figs.  7  to  I2.  It  will  be  seen  that  none  of  these  consti¬ 
tute  a  perfect  labyrinth,  but  on  closer  study  it  is  found  that  they 
all  possess  certain  features  which  are  characteristic  of  it.  In  the 
first  place,  that  which  was  transplanted  in  the  form  of  an  open 
auditory  cup  developed  after  the  operation  into  a  closed  vesicle 
containing  endolymph.  This  did  not  then  remain  a  simple  vesicle, 
but  exhibited  the  tendency  to  subdivision  into  two  or  more  com¬ 
partments,  the  utriculus  and  sacculus,  as  seen  in  Figs.  7, 8, 12.  In  the 
walls  ofthese  compartments  there  are  areas  of  specialized  epithelium 
representing  the  maculae  acusticae.  In  Fig.  7  there  opens  out  of 
the  more  dorsal  compartment  a  distinct  endolymphatic  appendage. 
A  typical  semicircular  canal  is  not  present  in  any  of  them;  but 
what  may  be  called  a  canal  tendency  is  seen  in  Fig.  8,  where 
there  is  a  tube  uniting  the  two  principal  compartments.  The 
small  blind  pouches  leading  off  the  main  vestibule,  three  of  which 
are  present  in  Fig.  10,  doubtless  represent  abortive  canals.  In 
transverse  section  they  are  perfectly  round  and  look  like  typical 


556 


George  L.  Streeter 


canals.  It  may  be  recalled  that  Riidinger  (’88)  described  the 
semicircular  canals  as  developing  in  the  form  of  blind  tubes  sprout¬ 
ing  out  from  the  general  vesicle.  It  is  quite  possible  that  he  was 
dealingwith  an  abnormal  embryo  and  had  the  same  form  of  canals 
that  we  see  in  Fig.  lo. 

The  ear  vesicles  are  more  or  less  completely  enveloped  in  con¬ 
nective  tissue  membranes  and  they  are  partly  incorporated  in 
masses  of  cartilage,  some  of  which  belongs  to  the  normal  cartilagi¬ 
nous  cranium  and  some  of  it  is  the  regular  cartilaginous  capsule 
of  the  labyrinth,  the  two  fusing  together  in  some  places. 

In  four  cases  (Figs.  7,  8,  9  and  10)  a  group  of  ganglion  cells  and 
nerve  fibers  are  attached  to  the  median  side  of  the  vesicle  near  its 
caudal  end  and  extend  toward  the  central  nervous  system.  In 
one  instance  (Fig.  7)  the  nervous  connection  between  vesicle  and 
brain  at  the  junction  of  olfactory  lobe  and  fore-brain,  is  complete, 
though  it  is  only  a  few  fibers  that  actually  enter  the  brain.  As  the 
acoustic  ganglion  at  the  time  of  the  operation  is  attached  to  the 
auditory  cup  some  of  its  ganglion  cells  are  undoubtedly  carried 
along  with  it,  and  it  is  probable  that  it  is  these  cells  that  furnished 
the  nerve  connections  just  described.  At  the  time  the  transplanted 
ear  cup  was  slipped  into  its  pocket  the  adherent  ganglion  cells 
must  have  been  lodged  in  various  positions  as  regards  the  ear  cup 
and  the  fact  that  they  all  come  finally  to  lie  on  the  median  side  of 
the  vesicle  and  lead  toward  the  brain  must  be  explained  by  some 
theory  of  an  attraction  existing  between  brain  and  nerve. 

When  we  have  to  deal  with  a  transplanted  labyrinth  that  has 
reached  a  development  equal  to  those  that  function  in  young  tad¬ 
poles,  and  has  established  communication  with  the  central  ner¬ 
vous  system,  we  might  expect  that  it  would  show  some  sign  of 
physiological  activity.  The  failure  of  it  to  do  so  is  perhaps  best 
accounted  for  by  the  fact  that  the  point  of  entrance  into  the  brain 
is  so  fa,r  away  from  the  hind-brain  centers  and  the  spinal  cord  that 
connections  with  these  are  not  established.  If  the  experiments  were 
varied  and  the  vesicle  transplanted  to  some  point  in  the  neighbor¬ 
hood  of  the  occipital  nerves  this  difficulty  would  be  obviated. 


Experiments  on  the  Developing  Ear  V estcle 


557 


Figs.  7  to  12.  Graphic  reconstructions  showing  the  form  and  relations  developed  by  transplanted  ear 
vesicles,  one  to  three  months  after  the  operation.  In  all  six  cases  the  right  ear  vesicle  was  removed  and 
the  left  vesicle  transplanted  into  a  subdermal  pocket  between  eye  and  nostril.  In  Figs.  7,  8,  9  and  10 
the  acoustic  nerve  and  ganglion  extended  from  ear  vesicle  toward  brain;  in  Fig.  7  the  connection  was 
complete,  the  fibers  entering  at  junction  of  fore-brain  and  olfactory  lobe.  Central  nervous  system, 
shaded;  ear  vesicle,  solid  black. 


558 


George  L.  Streeter 
Conclusions 


,  In  the  tadpole  the  ear  vesicles  are  essential  for  the  development 
of  the  power  of  equilibration,  but  the  study  of  normal  specimens 
shows  that  well  developed  equilibration  may  be  present  before 
the  completion  of  the  semicircular  canals;  the  latter  as  such  are 
therefore  not  essential. 

When  both  vesicles  are  removed  no  other  organ  compensates 
for  their  loss  and  the  animal  is  completely  and  permanently  help¬ 
less  as  regards  the  maintenance  of  equilibrium.  When  only  one 
ear  vesicle  is  taken  out  the  remaining  vesicle  is  capable  of  perform¬ 
ing  the  work  of  both  so  perfectly,  that  the  casual  observer  would 
mistake  them  for  normal  individuals. 

Transplantation  of  the  ear  vesicle  shows  that  the  group  of  cells 
forming  the  auditory  cup  or  primitive  ear  vesicle  is  specialized  to 
that  degree  that  although  removed  from  their  natural  relations 
and  placed  in  a  new  environment  they  still  continue  to  differentiate 
themselves  into  a  structure  approximating  the  normal  labyrinth. 
A  nerve  and  ganglion  develops,  and  complete  nervous  connection 
may  be  established  between  the  transplanted  vesicle  and  the  brain 
at  an  abnormal  place.  Where  the  latter  occurred  it  did  not  give 
evidence  of  any  functional  ability. 


LITERATURE 

Lee,  F.  S.,  ’93. — A  Study  of  the  Sense  of  Equilibrium  in  Fishes.  Journ.  of  Physiol., 
vol.  XV  and  xvii. 

’98. — The  Function  of  the  Ear  and  Lateral  Line  in  Fishes.  Amer.  Journ. 

.  of  Physiol.,  vol.  i. 

Lewis,  W.  H.,  ’04. — Experimental  Studies  on  the  Development  of  the  Eye  in 
Amphibia.  Amer.  Journ.  Anat.,  vol.  iii. 

Lyon,  E.  P.,  ’00. — A  Contribution  to  the  Comparative  Physiology  of  Compensatory 
Motions.  Amer.  Journ.  of  Physiol.,  vol.  iii. 

Parker,  G.  H.,  ’05. — ^The  Skin,  Lateral-Line  Organs  and  Ear  as  Organs  of 
Equilibration.  Proceedings  of  Amer.  Zool.  Soc.,  Science,  vol.  xxi. 

Prentiss,  C.  W.,  "01. — The  Otocyst  of  Decapod  Crustacea;  its  Structure,  Develop¬ 
ment  and  Functions.  Bull.  Museum  Compar.  Zool.,  Harvard 
College,  vol.  xxxvi. 

Rudinger,  ’08. — Zur  Anatomie  u.  Entwickelungdes  inneren  Ohres.  Berlin,  1888. 


THE  JOURNAL  OF  EXPERIMENTAL  ZOOLOGY  is  issued 

^  ^  quarterly.  A  volume  consists  of  four  numbers,  containing  from  100 

to  200  pages  each,  with  numerous  illustrations. 

PRICE  OF  SUBSCRIPTION  PER  VOLUME 

(payable  in  advance) 

To  subscribers  in  the  United  States,  Canada  and  Mexico,  $5.00 
To  subscribers  in  other  countries  -  -  -  -  -  5.50 

Price  of  single  copies . 2.00 

Tt\ese  prices  are  net  and  under  no  condition  subject  to  discount 


Remittances  should  be  made  by  Postal  Money  Order  {Mandat  de 
Paste,  Poslanweisung)  or  by  draft  on  New  York,  payable  to  The 
Journal  of  Experimental  Zoology. 

Address  all  communications  to 

•  • 

THE  JOURNAL  OF  EXPERIMENTAL  ZOOLOGY, 
N.  E.  Cor.  Wolfe  and  Monument  Streets, 

Baltimore,  Md.,  U.  S.  A. 


